Single particle cryo-electron microscopy (cryo-EM) is emerging as powerful technique for visualizing structures of macromolecules without the need for large sample quantities often required for alternative techniques such as X-ray crystallography and NMR. Recent technological advances have helped to achieve near-atomic structure determination of macromolecules by cryo-EM. However, cryo-EM for structure determination of small (<200 kDa) macromolecules is quite limited. Cryo-EM image data are low contrast, and small particles often lack well-defined structural features required for the image alignment step of 3D reconstruction. Additionally, the method is technically challenging, low-throughput, and expensive-further hindering its widespread adoption. We propose to use DNA nanotechnology to develop a novel suite of tools to overcome the size and throughput limitations of cryo-EM. DNA nanotechnology allows us to create soluble nanostructures with an unprecedented combination of spatial resolution and chemical versatility. In principle, we can attach any moiety to our devices as long as it has a DNA binding domain or it can be coupled to DNA. First, we will design and optimize DNA frame nanostructures that will bind and orient small DNA binding proteins and serve as high-contrast fiducial markers for cryo-EM imaging and tomography. We will also construct DNA barcode nanostructures and attach them to the DNA frames for sample multiplexing. We will validate our methods by determining the structure of a well-characterized DNA-binding protein called TALE that has been previously crystalized. We will then work with our collaborators to determine first time the structure of nuclear receptor called LRH-1 that is known to play essential roles in stem cell differentiation, development, and many other vital cellular processes. Second, we will design and optimize DNA prism nanostructures to attach recombinant antigen-binding fragments (Fabs) that will bind and orient small proteins that do not naturally bind to DNA. To attach Fabs to DNA prisms, we will couple Fabs to DNA, which will then bind to DNA prisms. We will attach DNA barcodes to DNA prisms for sample multiplexing. We will validate our approach by determining the structure of a well- characterized green fluorescent protein variant called EGFP. This technology will hugely improve our ability to solve near-atomic resolution cryo-EM structures of small proteins in a high-throughput manner. We will apply our method to study proteins with relevance to human diseases, and expect that our efforts will ultimately enhance structure-based drug design efforts to combat those diseases.